摘要
目的探索和优化大鼠视网膜色素上皮(RPE)细胞分离培养的方法,评价RPE细胞的存活状态及细胞基质金属蛋白酶(MMP)的表达,为相关眼底疾病的研究提供细胞来源。方法采用改良的三步酶消化法分离大鼠RPE细胞,并进行细胞体外培养。倒置显微镜观察细胞形态,细胞生长曲线评价不同培养代数的RPE细胞的增殖活力。免疫荧光检测CRALBP和角蛋白表达鉴定RPE细胞,并观察不同培养代数RPE细胞中多种基质金属蛋白酶的表达。结果分离培养的RPE细胞可呈梭形、六角形,并维持RPE细胞特征性蛋白CRALBP和角蛋白表达,但细胞内色素成分随着细胞分裂和传代次数的增多逐渐减少。基质金属蛋白酶MMP2、MMP3、MMP9和MMP10在第1代和第3代RPE细胞中均表达阳性,且表达强度未见明显改变。结论应用改良的三步酶消化法可以成功的分离培养大鼠RPE细胞,并在第1代和第3代RPE细胞维持基质金属蛋白酶MMP2、MMP3、MMP9和MMP10的阳性表达。体外培养的大鼠RPE细胞为研究视网膜相关疾病提供了细胞模型。
Objective To improve the isolation and culture techniques of rat retinal pigment epithelial（RPE） cells and investigate the expression of matrix metalloproteinases（MMPs） in the cultured RPE cells.Methods The brown Norway（BN） rat RPE cells were isolated by a modified three-step enzyme digestion.The growth curves of the RPE cells of different passages were measured.The expression of marker protein CRALBP and cytokeratin,and MMPs in the RPE cells of different passages were observed by immunofluorescence microscopy.Results The primary cultured RPE cells showed a polygonal shape and contained a large amount of pigments.The pigment in the cells was gradually lost at later passages as a result of dilution by cell division.Immunofluorescence microscopy detected the positive expression of specific markers CRALBP and cytokeratin in the RPE cells.Positive expressions of MMP2,MMP3,MMP9 and MMP10 were found in passage 1 and 3 RPE cells.Conclusion A larger amount of RPE cells can be obtained by this modified three-step enzymatic digestion.The positively passaged MMP2-,MMP3-,MMP9-and MMP10-expressing RPE cells may become useful cell models for further studies of retinal diseases.
出处
《中国实验动物学报》
CAS
CSCD
2010年第2期109-112,I0002,I0003,共6页
Acta Laboratorium Animalis Scientia Sinica
基金
国家自然科学基金(NO.30672284)
中国博士后科学基金(20080430288)资助
关键词
视网膜色素上皮细胞
基质金属蛋白酶
细胞培养
大鼠
Retinal pigment epithelial cell
Matrix metalloproteinase
Enzyme digestion
Cell culture
Rat
